Specific binding of Bacillus thuringiensis Cry2A insecticidal proteins to a common site in the midgut of Helicoverpa species.
For a long time, it has been assumed that the mode of action of Cry2A toxins was unique and different from that of other three-domain Cry toxins due to their apparent nonspecific and unsaturable binding to an unlimited number of receptors. However, based on the homology of the tertiary structure among three-domain Cry toxins, similar modes of action for all of them are expected. To confirm this hypothesis, binding assays were carried out with (125)I-labeled Cry2Ab. Saturation assays showed that Cry2Ab binds in a specific and saturable manner to brush border membrane vesicles (BBMVs) of Helicoverpa armigera. Homologous-competition assays with (125)I-Cry2Ab demonstrated that this toxin binds with high affinity to binding sites in H. armigera and Helicoverpa zea midgut. Heterologous-competition assays showed a common binding site for three toxins belonging to the Cry2A family (Cry2Aa, Cry2Ab, and Cry2Ae), which is not shared by Cry1Ac. Estimation of K(d) (dissociation constant) values revealed that Cry2Ab had around 35-fold less affinity than Cry1Ac for BBMV binding sites in both insect species. Only minor differences were found regarding R(t) (concentration of binding sites) values. This study questions previous interpretations from other authors performing binding assays with Cry2A toxins and establishes the basis for the mode of action of Cry2A toxins.
Hernández-Rodríguez
Carmen Sara
CS
Departamento de Genética, Facultad de CC. Biológicas, Universidad de Valencia Valencia, Spain.
Van Vliet
Adri
A
Bautsoens
Nadine
N
Van Rie
Jeroen
J
Ferré
Juan
J
D000818:Animals; D001413:Bacillus thuringiensis / Q000502:physiology*; D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins / Q000378:metabolism*; D004731:Endotoxins / Q000378:metabolism*; D041981:Gastrointestinal Tract / Q000187:drug effects*; D006460:Hemolysin Proteins / Q000378:metabolism*; D007457:Iodine Radioisotopes / Q000378:metabolism; D007700:Kinetics; D007915:Lepidoptera / Q000187:drug effects*; D011485:Protein Binding; D013194:Staining and Labeling
D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins; D004731:Endotoxins; D006460:Hemolysin Proteins; D007457:Iodine Radioisotopes; C030956:insecticidal crystal protein, Bacillus Thuringiensis